The sexually transmitted disease gonorrhea has remained in the human population for thousands of years, owing in part to the inability of infected individuals to develop long-term immunity to the bacterium that causes this disease, Neisseria gonorrhoeae (Gc). Gc evades immune system surveillance by constantly changing its arsenal of surface-exposed antigens. Changes in pilin, the major subunit of type IV pili, arise from a DNA recombination-based process called antigenic variation (Av). In pilin Av, new DNA sequence is transferred into the pilin expression locus, pilE, from incomplete pilin copies (pilS) found elsewhere in the Gc chromosome. Although a great deal is currently understood about the mechanism of pilin Av, the capacity of Gc to undergo this process upon encountering host cells has not previously been investigated. Therefore, the importance of pilin Av in progession of gonorrheal disease remains unknown. The goal of this proposal is to examine pilin Av in Gc during infection of polarized epithelial cells and peripheral blood neutrophils (PMN), both of which play key roles in disease pathogenesis. First, I will establish parameters for infection of epithelial cells and PMN by Gc, with a focus on determining whether type IV pili are necessary for adherence, internalization, survival, and exit of Gc from these cells. Second, I will calculate the frequency of pilin Av for Gc encountering epithelial cells and PMN. Finally, I will use a genetic approach to examine whether pilin Av is essential to the ability of Gc to reside within epithelial cells and PMN or to cross-polarized epithelial monolayers. The results obtained from these studies will provide crucial insight into how pilin Av contributes to the ability of this pathogen to establish productive infections in host cells.